Rapid detection of Salmonella enteritidis by PCR amplification of the SefA gene and it's cloning.

The emergence of Salmonella enteritidis as an important food-borne pathogenesis in humans, demands the development of novel detection and intervention strategies. It is generally accepted that fimbriae are an important factor in bacterial survival and persistence in the host. This study is directed towards the method of amplifying and cloning the SefA gene, which encode Salmonella enteritidis fimbrial protein. Strains used for these studies were S. enteritidis (E3), which were collected from Kermanshah region. Chromosomal DNA was extracted by boiling method and PCR reaction was performed and single band of 511 bp amplified by SefA-F and SefA-R primers. The resulting PCR product was inserted into the cloning vector (pTZ57R/T). In order to amplify the recombinant plasmid, E. coli DH5 alpha bacteria were transformed with SefA-pTZ57R/T. Recombinant clones were identified by blue/white selection and purified recombinant plasmids were indicated by an alkaline lysis procedure. Identity of the SefA-pTZ57R/T product was confirmed by RFLP and sequencing. Nucleotide and protein alignment with BLAST software showed that the sequence of the SefA gene derived from S. enteritidis (E3), which was cloned in the pTZ57R/T vector, was 99% identical to that of the Genbank (L11008). The sequence of the SefA gene from S. enteritidis (E3) differed only in two nucleotides and one amino acid. The cloned SefA gene from S. enteritidis (E3) was submitted to the NCBI Genbank (EF553334).

Rapid detection of Potato Y potyvirus in potato farms of Kermanshah using RT-PCR amplification of the P1-protease gene and its cloning.

In this study, the RT-PCR method was used to detect the Y virus in potato tubers and leaves. Samples suspicious of virus infection with symptoms of virus infection were gathered from farms in Kermanshah and placed in plastic bags and kept at -80 degrees C temperature in order to maintain the RNA of the virus until extraction. The extraction and purification of RNA were carried out using Tri-Reagent kit. One of the virus genes is the P1 protease gene which codes a proteinase enzyme. This enzyme plays a role in breaking the initial polyprotein. For amplification of this gene three primer, including primer-1, primer-2 and primer-3, were designed and used. Using primer 1 and reverse transcriptase enzyme, cDNA was synthesized and then PCR was performed using the primers 1, 2 and 3. The PCR products were examined by agarose gel electrophoresis (1%). Consequently, two pieces of DNA (400 and 800 bp) were yielded which were identical to the genome DNA sequencing. Thus, the proposed technique is a convenient method for quick and accurate detection of viruses and, therefore, the application of this method for detecting Potato Y virus in potato farms is recommended.

Rapid detection of Salmonella dublin by PCR amplification of the SopE gene and its cloning.

This study is directed towards the method of amplifying and cloning the SopE gene, that encodes Salmonella outer protein E. Strains used in this study were S. dublin collected from Kermanshah province. Genomic DNA was extracted by the general boiling method. Using the specific primers, a part of SopE gene was multiplied. The PCR product was inserted into the cloning vector (pTZ57R/T). Furthermore, E. coli DH5alpha bacteria were transformed to amplify the recombinant plasmid. Recombinant clones were identified by blue/white selection. Recombinant plasmids were purified by alkaline lysis procedure. Moreover, identity of the SopE/pTZ57R/T product was confirmed by restriction enzyme digestion assay and sequencing. Finally, the cloned gene was compared with that published by the NCBI Genbank (L78932). The results showed that the obtained sequence differed in four nucleotides which resulted in two amino acid differences. The cloned SopE was submitted to the NCBI Genbank (EU399750).

Molecular study of Salmonella enteritidis in poultry samples by PCR, plasmid curing, antibiotic resistance and protein pattern analysis.

In this study two control isolates of Salmonella enteritidis, RTCC1623 and RTCC1624, were obtained from the institute ofRazi (Karaj-Iran) and 14 strains were isolated from poultry samples in Kermanshah province of Iran, according to a standard protocol. These isolates were confirmed by PCR amplification of SefA gene fragments. Results showed that, 6 isolates of 14 isolates of Salmonella which their biochemical tests were positive contain 511 bp amplified fragments of the SefA gene. In other purpose, to correlating the presence of plasmids with antibiotic resistance and protein pattern, plasmid DNA was isolated before and after plasmid curing by using the alkaline lysis method. Strains of S. enteritidis contain seven different plasmid profiles (P1-P7) which were characterized by antibiotic resistance and protein pattern. Our observed showed, there was a high molecular weight plasmid with Rf 0.17 in all strains and the frequency of other plasmids was low. The plasmid with Rf about 0.2 is responsible for resistance to Cephalothin and the isolates that lost it were susceptible to this antibiotic. All strains, 100%, were resistant to ampicillin before and after curing of strains. According to present findings, PCR is a rapid and sensitive method for typing of S. enteritidis and plasmid profiling; antibiotic resistance and protein pattern are suitable methods for subtyping of S. enteritidis isolates. No direct correlation was found between plasmid contents, antibiotic resistance patterns and protein profiles of local S. enteritidis isolates.

An RNA tertiary structure in the 3' untranslated region of enteroviruses is necessary for efficient replication.

RNA tertiary structures, such as pseudoknots, are known to be biologically significant in a number of virus systems. The 3' untranslated regions of the RNA genomes of all members of the Enterovirus genus of Picornaviridae exhibit a potential, pseudoknot-like, tertiary structure interaction of an unusual type. This is formed by base pairing between loop regions of two secondary structure domains. It is distinct from a potential, conventional pseudoknot, studied previously in poliovirus, which is less conserved phylogenetically. We have analyzed the tertiary structure feature in one enterovirus, coxsackievirus A9, using specific mutagenesis. A double mutant in which the potential interaction was destroyed was nonviable, and viability was restored by introducing compensating mutations, predicted to allow the interaction to reform. Phenotypic pseudorevertants of virus mutants, having mutations designed to disrupt the interaction, were all found to have acquired nucleotide changes which restored the potential interaction. Analysis of one mutant containing a single-base mutation indicated a greatly increased temperature sensitivity due to a step early in replication. The results show that, in addition to secondary structures, tertiary RNA structural interactions can play an important role in the biology of picornaviruses.